1. Field of the Invention
The present invention relates to a laser scanning microscope.
2. Description of the Related Art
The laser scanning microscope enables obtaining two-dimensional cross-sectional images by optically slicing, but not damaging, a sample such as living cell, tissue, or the like, and obtaining a three-dimensional image from a plurality of cross-sectional images.
The laser scanning microscope observing a biological sample irradiates fluorescent reagents or fluorescent protein introduced into the sample with a laser beam, and measures the fluorescence therefrom to be imaged. At that time, a plurality of fluorescent reagents or fluorescent proteins are introduced into the sample, and it is possible to observe a plurality of chemical substances in the cells. In order to excite these plurality of fluorescent materials, an excitation laser beam having a plurality of wavelengths is required. An example of a laser beam source for outputting an excitation laser beam having a plurality of wavelengths is shown in FIG. 1.
On the other hand, a fading phenomenon that an amount of fluorescence from the fluorescent materials decreases accompanying an irradiation time of an excitation light is brought about. Therefore, it is necessary to prevent fading as much as possible by cutting off an unnecessary excitation light from or reducing light to the laser scanning microscope.
In order to achieve these objects, it has been proposed that an acousto-optic element (AOTF, AOF, AOM, or the like), or an electro-optic element (EOM) is utilized (for example, refer to FIG. 2 in AKIS P. GOUTZOULIS and DENNIS R. PAPE DESIGN AND FABRICATION OF ACOUSTO-OPTIC DEVICES, p. 246-258 (1944).
When an ultrasonic wave propagates in a solid body or a liquid, a periodic fluctuation in a refractive index for the light is brought about in parallel with the traveling direction of a sound and with the wavelength of the sound being as a period, by a photoelastic effect in the medium thereof. When a light is incident into the medium, part of the incident light is diffracted by an ultrasonic wave. This phenomenon is called an acousto-optic effect. The principle of operation of an acousto-optic element (AOM, AOTF, AOD, or the like) using this acousto-optic effect is shown in FIG. 2. When a high frequency (RF) voltage is applied to an optical crystal such as LiNbO3, PbMoO4 or TeO2 serving as a medium by attaching a transducer such as a piezoelectric body which transmits ultrasonic waves (RF), a high frequency acoustic wave is generated in the crystal. It is possible to control a transmitted light and a reflected light by utilizing a periodic variation in a refractive index due to the acoustic wave.
On the other hand, a phenomenon that the refractive index of the material is varied when a voltage is applied to a material is called an electro-optic effect. The phase of a light passing through in the material (a crystal such as LiNbO3, KDP, or ADP) is varied by applying a voltage, thereby making it possible to carry out amplitude modulation and phase modulation. One for which such an electro-optic effect is utilized is called an electro-optic element (EOM or the like).
Examples of the use of an acousto-optic element in a laser scanning microscope are shown in FIGS. 3 and 4.
FIG. 3 is a diagram showing an example in which an acousto-optic element 130 is used as high-speed selection (switching) means and lighting control (emission power control mechanism) means for a laser wavelength, and pulse shaping means and shutter means which utilize the switching function thereof. The details of the laser scanning microscope according to the prior art is shown in Jpn. Pat. Appln. KOKAI Publication No. 2000-206415. A plurality of lasers of shorter wavelengths of Ar or ArKr 110a, HeNe 110b and 110c are installed, and the emitted beams of those lasers are synthesized into one beam by a dichroic mirror or the like so as to be incident into the acousto-optic element 130. The acousto-optic element 130 enables wavelength splitting due to the RF frequency control, emission power due to the RF amplitude (voltage) control, and pulse-shaping of a continuous oscillation laser due to the RF-ON/OFF switching control at a high-speed.
FIG. 4 is a diagram showing an example in which a pulse laser which can change wavelengths is used as a laser beam source 110. The acousto-optic element 130 emits a laser of the selected wavelength by controlling an RF frequency in accordance with a selected wavelength of the laser beam source, and enables power control of an emitted beam due to the RF amplitude (voltage) control.
However, a crystal (LiNbO3, PbMoO4 or TeO2) used for the acousto-optic element 130 has the defect that the wavelength dispersion (group velocity delay dispersion; GDD) is extremely great, and the pulse width of a beam after being emitted is spread by the acousto-optic element 130. In a multiphoton excitation laser scanning microscope, the fluorescent luminous intensity is inversely proportional to a pulse width in a case of two photon excitation, and to the square of a pulse width in a case of three photon excitation, which has an extremely large influence.
As a countermeasure against pulse width spreading of an emitted beam due to the wavelength dispersion (GDD) of the acousto-optic element 130, means in PCT National Publication No. 10-512959 is shown in FIG. 5. This is configured such that a pre-chirping optical system 160 is inserted between the pulse laser beam source 110 and the acousto-optic element 130, and spreading of the pulse width after emission of the acousto-optic element 130 or on a plane of a specimen to be observed is offset by carrying out inverse dispersion (pre-chirping) of an amount of the wavelength dispersion (GDD) of the entire optical system up to the time of reaching the acousto-optic element 130 or the specimen to be observed after emission of a laser.
FIG. 6 is a schematic diagram of a typical pre-chirping optical system using prisms. A beam split by a first prism 161 in FIG. 6 is inversely-dispersed by a second prism 162 set to a difference between optical path lengths corresponding to an amount of wavelength dispersion (a divergence in a transmission velocity in a medium by a wavelength within a laser beam band) to pass through the first prism 161, and is made to return to the inside of the microscope illumination optical system, and therefore, the wavelength dispersion inside the illumination optical system can be offset.
More specifically, an amount of compensating dispersion in each wavelength is adjusted by adjusting an interval between the first and second prisms 161 and 162, thereby compensating the group velocity delay dispersion in the entire optical system from the pulse laser beam source 110 to an objective lens 320 of the fluorescent microscope body. At that time, the optical axis is shifted by adjusting the optical path length, and the angle of the optical axis is fluctuated due to a variation in the wavelength. Therefore, the position and the angle of the optical axis of an ultra-short pulse laser beam L to be emitted are adjusted by adjusting the positions and the angles of the prisms 161 and 162, and the mirror 163 as shown by the arrows.
A mirror 165 is shown in FIG. 6. The mirror 165 reflects the ultra-short pulse laser beam L emitted from the pulse laser beam source 110 so as to be orientated to the pre-chirping optical system 160, and is positioned at a position out of the optical axis of the ultra-short pulse laser beam L outputted from the pre-chirping optical system 160.
Next, pulse compression means utilizing an amount of wavelength dispersion (GDD) of the acousto-optic element described in Jpn. Pat. Appln. KOKAI Publication No. 2000-206415 will be described on the basis of FIG. 7. In a laser microscope which uses a pulse laser as the laser beam source 110, and which has an illumination optical system used for introducing an optical fiber 185 into a microscope, it is necessary to sufficiently spread the pulse width and sufficiently decrease the peak power at the incident side of the fiber by a pulse stretcher 180 such as a pre-chirping (inverse dispersion) optical system shown above in order to avoid linear and non-linear dispersive effect which the optical fiber 185 has. However, in a multiphoton excitation laser microscope, the pulse width must be sufficiently narrow in order to obtain a necessary fluorescent luminous intensity as shown above, and the photon density on the plane of the specimen is low in a beam whose pulse width has been spread after passing through a pulse stretcher and a fiber, which makes multiphoton excitation impossible. Then, an optical member having a great amount of wavelength dispersion (GDD), such as, for example, ZnSe, is installed at the emission end of the fiber, and a necessary pulse is obtained by compressing the pulse width. The acousto-optic element as well, as described above, can be used as a pulse compressor in the same way because an amount of wavelength dispersion (GDD) thereof is sufficiently great. Here, the advantage in utilizing the acousto-optic element is in the point that it can be utilized as wavelength splitting means, lighting control (power adjustment) means, and switching means as well.
The acousto-optic element has been mainly described above as the prior art. However, an electro-optic element as well can be applied to a laser scanning microscope in the same way.